EP1639012A2 - Suppression du rejet des transplants - Google Patents

Suppression du rejet des transplants

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Publication number
EP1639012A2
EP1639012A2 EP04743000A EP04743000A EP1639012A2 EP 1639012 A2 EP1639012 A2 EP 1639012A2 EP 04743000 A EP04743000 A EP 04743000A EP 04743000 A EP04743000 A EP 04743000A EP 1639012 A2 EP1639012 A2 EP 1639012A2
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EP
European Patent Office
Prior art keywords
animal
antibody
cells
regulatory
lymphocytes
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP04743000A
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German (de)
English (en)
Inventor
Andrew Nuffield Department of Surgery BUSHELL
Kathryn Nuffield Department of Surgery WOOD
Mahzuz Nuffield Department of Surgery KARIM
Vanessa Nuffield Department of Surgery OLIVEIRA
Brigit Nuffield Department of Surgery SAWITZKI
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Oxford University Innovation Ltd
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Oxford University Innovation Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2812—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • This invention relates to the suppression of rejection of transplants in animals.
  • Transplantation is the treatment of choice for end stage kidney, heart, liver and pancreas organ failure and despite considerable advances in the management of transplant rejection in recent years the vast majority of transplants are eventually rejected.
  • the current immunosuppressive regimens which depend on continual drug therapy predispose transplant patients to increased susceptibility to infections and cancer because even the most sophisticated drugs are unable to inhibit just those responses directed toward the transplant.
  • opportunistic infection remains one of the main causes of mortality in heart transplant patients and predictive calculations have shown that 30 years of continual immunosuppression carries a 100% risk of some types of cancer.
  • CD4+ T-helper lymphocytes are cells of the immune system and in normal situations play an essential role in immune responses that protect us from pathogenic organisms such as bacteria and viruses. In the context of transplantation however, these same cells are largely responsible for the rejection of organ transplants. It is widely known that rejection responses can be attenuated by administration of immunosuppressive agents, including anti-CD4 antibody which targets CD4+ T cells, but in recent years it has been shown that such antibody therapy can lead to the generation of sub- populations of T cells with the capacity to control or regulate destructive rejection responses. It is believed that regulatory cells arise in such situations because the presence of the anti-CD4 antibody prevents full T cell activation and the cells default to a regulatory or suppressive phenotype.
  • EP-A-0 240 344 describes the use of a monoclonal antibody directed against the CD4 antigen on helper T lymphocytes for the manufacture of a medicament for the treatment of a mammal to induce tolerance in the said mammal to a primary antigen, the treatment comprising administering to the subject mammal sufficient of the medicament to deplete significantly the population of T-helper lymphocytes in the subject mammal, challenging the subject mammal with the primary antigen and allowing the population of T-helper lymphocytes in the subject mammal to re-establish itself in the presence of the primary antigen so that tolerance to the primary antigen is established.
  • EP-A-0 474 691 describes how non-depleting CD4 antibodies, optionally together with CD8 antibodies, can produce tolerance to foreign immunoglobulins, bone marrow and skin grafts. Specifically a state of immunological tolerance to an antigen can be induced by the administration of these antibodies in the presence of said antigen.
  • antigens are usually foreign cellular antigens, but tolerance to soluble non-cellular antigens such as albumin or human gamma globulin (HGG) ' has also been achieved by intravenous administration to mice under the cover of anti-CD4 antibody.
  • lymphocytes with suppressive capacity were first described over thirty years ago 1 , but in recent years there has been renewed interest in the identification and characterisation of such regulatory T cells (T-reg).
  • T-reg regulatory T cells
  • Several cell surface markers have been identified that enrich for regulatory activity, one of which is CD25, the ⁇ subunit of the IL-2 receptor.
  • CD25 + CD4 + T-reg with the capacity to regulate responses in vitro have been identified in both mice 2"6 and humans 7"12 .
  • T-reg can suppress the proliferation and/or effector activity of both CD4 + 2 ' 4 and CD8 + 3 ' 5 ' 13 ' 14 T cells, can prevent the development of autoimmune disease " , and have been shown to play a role in both tumour immunity 18 ' 19 and transplantation 13,20"24 .
  • regulatory activity can be dependent on IL-10 25 , TGF- ⁇ 26 , and CTLA-4 26,27 .
  • An object of the present invention is to harness the potential of regulatory T cells in the suppression of transplant rejection and, in particular, to provide a method for the suppression of transplant rejection in an animal in which the disadvantages referred to above are alleviated or eliminated.
  • the present invention provides a method of suppressing rejection of an organ or tissue transplant in an animal comprising the following steps:
  • ICAM-1 ICAM-1, and a non-cellular protein antigen to generate a population of regulatory T- lymphocytes
  • the method further comprises administering to the animal a population of regulatory T-lymphocytes produced according to an ex vivo method comprising culturing T cells with an antibody directed at a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA-1, CD80, CD86 and ICAM-1, in the presence of cells that present either alloantigen or a non-cellular protein antigen.
  • a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA-1, CD80, CD86 and ICAM-1
  • the present invention provides the use of an antibody directed at a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA-1, CD80, CD86 and ICAM-1, for the manufacture of a medicament for the suppression of rejection of an organ or tissue transplant in an animal by a method which comprises administering the antibody to the animal together with a non-cellular protein antigen to generate in the animal a population of regulatory T-lymphocytes; reactivating said population of regulatory T-lymphocytes by further administration to the animal of the non-cellular protein antigen; and transplanting said organ or tissue whilst said population of regulatory T-lymphocytes is activated.
  • a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA-1, CD80, CD86 and ICAM-1
  • the present invention provides the use of a non-cellular protein antigen for the manufacture of a medicament for the suppression of rejection of an organ or tissue transplant in an animal by a method which comprises administering an antibody directed at a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA-1, CD80, CD86 and ICAM-1, to the animal together with the non-cellular protein antigen to generate in the animal a population of regulatory T- lymphocytes; reactivating said population of regulatory T-lymphocytes by further administration to the animal of the non-cellular protein antigen; and transplanting said organ or tissue whilst said population of regulatory T-lymphocytes is activated.
  • a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA-1, CD80, CD86 and ICAM-1
  • the invention further provides a method of treating a condition in an animal mediated by an immune response which comprises administering to said animal an antibody directed at a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA-1, CD80, CD86 and ICAM-1, and a non-cellular protein antigen to generate a population of regulatory T-lymphocytes which are then re-activated by subsequent administration of the original non-cellular antigen.
  • a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA-1, CD80, CD86 and ICAM-1
  • the method further comprises administering to the animal a population of regulatory T-lymphocytes produced according to an e vivo method comprising culturing T cells with an antibody directed at a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA-1, CD80, CD86 and ICAM-1, in the presence of cells that present either alloantigen or a non-cellular protein antigen.
  • a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA-1, CD80, CD86 and ICAM-1
  • Another aspect of the invention provides the use of an antibody directed at a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA-1, CD80, CD86 and ICAM-1, for the manufacture of a medicament for the treatment of a condition in an animal mediated by an immune response by a method which comprises admi stering the antibody to the animal together with a non-cellular protein antigen to generate in the animal a population of regulatory T-lymphocytes which are then reactivated by subsequent administration of the original non-cellular antigen.
  • a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA-1, CD80, CD86 and ICAM-1
  • a further aspect of the invention provides use of a non-cellular protein antigen for the manufacture of a medicament for the treatment of a condition in an animal mediated by an immune response by a method which comprises administering the non-cellular protein antigen to the animal together with an antibody directed at a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA-1, CD80, CD86 and ICAM-1, to generate in the animal a population of regulatory T-lymphocytes which are then re-activated by subsequent administration of the original non-cellular antigen.
  • the invention further provides an ex vivo method for generating a population of regulatory T lymphocytes comprising culturing T cells with an antibody directed at a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA-1, CD80, CD86 and ICAM-1, in the presence of cells that present either alloantigen or a non-cellular protein antigen.
  • a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA-1, CD80, CD86 and ICAM-1
  • Another aspect of the invention relates to a method of suppressing rejection of an organ or tissue transplant in a recipient animal comprising the following steps: (a) taking a sample ofT cells from the recipient animal;
  • Figure 1 shows that allo antigen-induced CD25 + CD4 + cells generated in vivo can regulate skin allograft rejection
  • Figure 2 shows that cells generated by culturing CD4 + T cells with cells presenting alloantigen in vitro can regulate skin allograft rejection and that the regulatory activity can be further enriched by sorting CD62L + CD25+CD4 cells generated by culturing
  • CD4 + T cells with cells presenting alloantigen in vitro show that activated CD25 + CD4 + cells generated against unrelated antigen in vivo can regulate skin allograft rejection;
  • Figure 4 shows that HGG re-challenge after 177/HGG pre-treatment in vivo leads to
  • Figure 5 shows that when combined with a single dose of anti-CD8 antibody, the anti- CD4 HGG + re-boost protocol leads to prolonged cardiac allograft survival in primary recipients;
  • Figure 6 shows proposed models for regulation of allograft rejection by regulatory cells.
  • regulatory T cells are generated by in vivo exposure to a non-cellular protein antigen in the presence of an antibody directed at a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA- 1, CD80, CD86 and ICAM-1, preferably an anti-CD4 antibody.
  • a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA- 1, CD80, CD86 and ICAM-1, preferably an anti-CD4 antibody.
  • These T-reg are then re-activated by a second exposure to the non-cellular protein antigen prior to transplantation taking place. Once reactivated, the T-reg acquire the capacity to control the activity of graft destructive T cells so that transplant rejection can be suppressed or prevented provided that transplantation takes place whilst the T-reg are activated.
  • T-reg T-reg
  • T cells can by enriched by sorting T cells that are CD4+ and express CD25, the ⁇ chain of the interleukin 2 receptor (IL-2R), and/or CD62L or other appropriate markers, and have been shown to express the transcription factor Foxp3.
  • IL-2R interleukin 2 receptor
  • CD62L CD62L or other appropriate markers
  • T-reg by non-cellular protein antigen plus therapy with an antibody directed at a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA-1, CD80, CD86 and ICAM-1, preferably anti-CD4 antibody therapy, followed by reactivation with the same protein allows them to regulate non-specifically to alloantigen in vivo.
  • a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA-1, CD80, CD86 and ICAM-1, preferably anti-CD4 antibody therapy, followed by reactivation with the same protein allows them to regulate non-specifically to alloantigen in vivo.
  • T-reg generated by administration of a non-cellular protein antigen such as human gamma globulin (HGG) combined with an antibody directed at a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA-1, CD80, CD86 and ICAM-1, preferably anti-CD4 antibody, can prevent the rejection of grafts such as skin allografts. This is extremely attractive in view of the fact that the potential for the clinical transmission of infectious agents will limit the feasibility of the administration of human products such as blood.
  • Clinical protocols are possible according to the present invention in which patients awaiting transplantation are given a well-defined, quality-controlled non-cellular antigen combined with immunotherapy with an antibody directed at a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA-1, CD80, CD86 and ICAM-1, preferably anti-CD4 antibody immunotherapy, to generate T-reg.
  • a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA-1, CD80, CD86 and ICAM-1, preferably anti-CD4 antibody immunotherapy, to generate T-reg.
  • T cells would be maintained by routine antigen re-challenge then re-activated immediately prior to transplantation. Under the correct circumstances these cells will be capable of regulating responses against the graft.
  • regulatory T cells are generated ex vivo by culturing T cells with an antibody directed at a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA-1, CD80, CD86 and ICAM-1, in the presence of cells that present either alloantigen or a non-cellular protein antigen.
  • the T cells may be taken from a recipient animal or patient.
  • the resulting population of regulatory T cells can be introduced into the patient for use in prevention of transplant rejection or for treating autoimmune disease or graft-versus-host disease.
  • the alloantigen may comprise cells taken from a donor animal or cells pulsed with antigen taken from a donor animal, wherein the donor animal may be the source of the transplanted organ or tissue.
  • regulatory T cells could be generated in vivo in a patient using the non-cellular protein antigen approach, and this could be followed post- transplant by treatment with regulatory T cells that have been generated ex vivo by using cells from the donor (whose identity would of course be known post-transplant) to activate T cells from the recipient patient in the presence of an antibody directed at a cell surface antigen selected from the group consisting of CD4, CD8, CD 154, LFA-1, CD80, CD86 and ICAM-1.
  • T-regs generated by the 177/HGG - HGG re-challenge protocol have the ability to regulate rejection of BIO skin allografts suggesting that they might operate either by cross-reactivity or bystander regulation.
  • Cross-reactivity seems the least likely of these two possibilities since it is presumed that these self-restricted CD25 + CD4 + T cells will have been generated in response to HGG peptides that are completely unrelated to alloantigen.
  • the probability is that such cells act via bystander regulation in which activated T-reg regulate responses in a restricted local microenvironment, mediated by cytokines such as IL- 10 24 ( Figure 3c) and IFN- ⁇ ( Figure 4).
  • T- reg populations are generated that are specific for each of the alloantigens expressed by the graft, rejection is prevented.
  • T-reg populations specific for a single donor antigen undergo activation by the graft, they are also able to overcome rejection through the process of bystander regulation.
  • Prevention of graft rejection by T-reg that have been generated against non-graft antigens is possible only when the regulatory cells are first activated by deliberate antigen re-exposure.
  • the duration of graft protection offered by these non-graft-specific T-reg is likely to be fairly short-lived since these cells are unlikely to remain activated for a significant length of time in the absence of specific antigen stimulation 40 ; however, it is probable that this period of protection allows operational tolerance to the graft to develop by other mechanisms 41 .
  • the ability to generate T-reg populations by controlled exposure to defined antigens has important implications for clinical transplantation and may also have implications for autoimmune disease where attenuation of immune responses is also an important goal.
  • the present invention can be used in suppressing the rejection of allogeneic organs, tissues or cells of any type in an animal but is particularly applicable to the suppression of transplant rejection in humans.
  • the CD4 molecule expresses several different epitopes which in turn can lead to the production of different anti-CD4 antibodies after immunisation. All such anti-CD4 antibodies are capable of binding CD4 but they will have a range of properties based on affinity and isotype. For example, rat anti-mouse IgG2b antibodies deplete CD4+ T cells whereas broadly speaking IgG2a antibodies do not deplete. It has been shown that both depleting and non-depleting anti-CD4 antibodies can induce tolerance to an antigen and both can be used according to the present invention. However, non- depleting anti-CD4 antibodies may be preferred for use in clinical protocols since depletion of CD4+ T-cells maybe difficult to control and long lasting.
  • any particular anti-CD4 antibody for use according to the invention may be confirmed by the ability of the antibody to induce tolerance to a soluble non- cellular protein antigen such as HGG using the ELISA assay shown in Figure 3a.
  • cell surface antigens may also be suitable targets for this type of approach including CD8, CD154 and LFA-1 on T cells and CD80, CD86 and ICAM-1 on antigen presenting cells.
  • CD8 CD154 and LFA-1 on T cells and CD80
  • CD86 CD86 and ICAM-1 on antigen presenting cells.
  • the ability of antibodies against such candidate molecules to induce tolerance to non-cellular protein antigens can also be determined by ELISA assays similar to that in Figure 3 a.
  • Antibodies against the CD4 antigen and other cell surface molecules can be generated by standard methods involving the fusion of antibody secreting B cells with cell lines selected for their ability to confer in vitro immortality on the antibody secreting cells.
  • DNA encoding monoclonal antibodies, antigen binding chains or domains can be cloned and expressed using standard methods of recombinant DNA technology.
  • Recombinant antigen binding molecules can be manipulated to improve therapeutic properties such as specificity, affinity, half-life and lack of immunogenicity.
  • the use of antibodies generated in rodents or other non-human animals in therapy in man is limited by the reaction of the patient's immune system to these antibodies.
  • anti-CD4 antibodies induce tolerance to themselves and the number of times that the anti-CD4 antibody needs to be administered when used according to the invention is limited, the anti-globulin response elicited by use of a rodent or other non- human antibody should be minimal.
  • rodent e.g. rat or mouse
  • non-human animal e.g. horse
  • the anti-CD4 antibody has been engineered to limit the anti-globulin response.
  • antibodies engineered in this way are chimeric antibodies (where the constant regions of a non-human antibody are replaced by human constant regions) and humanised antibodies where the antibody is engineered to appear human to the immune system of the recipient.
  • humanised antibodies are CDR-grafted antibodies where as well as replacing the constant regions of a non-human antibody with a human constant region, the framework regions of the variable regions are also replaced by human variable regions. Production of a humanised anti-CD4 antibody is described, for example, in WO-A-92 05274.
  • the antibody may be administered intravenously by injection or infusion; intraperitoneal infusion is also possible.
  • the antibody may be formulated for administration to humans in a standard manner, generally together with at least one physiologically acceptable carrier.
  • the antibody will generally be formulated in solution in a physiologically acceptable carrier optionally with one or more other ingredients.
  • Preferably the antibody is formulated in sterile isotonic buffered saline.
  • the non-cellular protein antigen is also generally administered parenterally, by intravenous infusion for the generation of a population of regulatory T cells in vivo. If a depot effect is required the non-cellular protein may be delivered by an intramuscular route.
  • the antibody preferably anti-CD4 antibody
  • the antibody is administered to the subject in a dose which is clinically effective to induce tolerance to an antigen in that subject. In any particular case, the precise dose will be at the discretion of the attendant physician but will generally be in the range 0.25 to 25mg/kg. Generally from 1 to 5 doses but preferably 2 or 3 doses of the antibody, preferably anti-CD4 antibody, are given over a period of 2-5 days.
  • the non-cellular protein antigen will be given concomitantly with the antibody to generate a population of regulatory T-lymphocytes which will then be expanded and/or maintained by repeated administration of the non-cellular antigen alone.
  • the minimum number of doses of the antigen will be two but maintenance of the regulatory population may require 10 or more doses in total.
  • Suitable non-cellular protein antigens for use in accordance with the present invention should have the following characteristics: (i) they should be immunogenic, i.e. must be a protein to which humans are not naturally tolerant;
  • the protein must be physiologically acceptable and non-toxic at the levels used.
  • administration of the tolerogenic protein with the immunomodulatory antibody should carry a minimal risk of sensitization. This may be assessed by established in vitro assays for the presence of circulating antibody.
  • non-cellular soluble protein antigen Before any individual non-cellular soluble protein antigen is used in the method according to the present invention, it would need to have received regulatory approval for clinical use and proteins are preferred which have already received such approval.
  • suitable non-cellular soluble protein antigens include human gamma globulin, equine gamma globulin and ovalbumin.
  • the animal is treated with additional immunosuppression or adjunctive therapy to attenuate any immediate rejection response that occurs.
  • the additional immunosuppression or adjunctive therapy may comprise administration of a sub-therapeutic dose of an immunosuppressive agent, preferably an agent used in a manner (time/dose) that does not block the function of the regulatory T cells, in the immediate post-operative period.
  • Suitable immunosuppressive agents or adjunctive therapies include treatment with an anti-CD8 antibody or with rapamycin. The intention is that the combination of the antibody plus non-cellular protein antigen treatment with a sub-therapeutic dose of an immunosuppressive agent would lead leads to the prolonged survival of fully allogeneic cardiac allografts in fully immunocompetent recipients.
  • a sub-therapeutic dose can be identified by reference to clinical studies identifying suitable therapeutic doses.
  • T cells can be sub-divided into CD4 + and CD8 + populations and both can play non-overlapping roles in graft rejection in intact animals.
  • the anti-CD4/HGG + HGG re-challenge protocol targets only CD4 + T cells.
  • the alloantigen used in the ex vivo method may be cells from a donor animal or cells pulsed with antigen.
  • the cells could be isolated from peripheral blood and in the case of a cadaveric donor the cells could either be isolated from the spleen or from donor peripheral blood. If the antigen of interest has not been identified, cells can be pulsed with a mixture of antigens obtained from pancreatic cells of the donor animal which is likely to include the antigen of interest.
  • T cells generated ex vivo by exposing CD4 + T cells in culture to cells presenting donor alloantigen or an unrelated protein antigen in the presence of an antibody directed at a cell surface antigen selected from the group consisting of CD4, CD8, GDI 54, LFA-1, CD80, CD86 and ICAM-1, preferably an anti-CD4 antibody, could be used either as an alternative or complementary therapeutic approach to prevent or treat transplant rejection.
  • T cells generated in the cultures would then be administered intravenously to the animal either before or after transplantation ( Figure 2a).
  • T cells with regulatory activity generated in the cultures can be further enriched by purifying cells express CD62L, i.e. CD62L + CD25 + CD4 + T cells ( Figure 2b).
  • the present invention also provides a method of treating a condition in an animal mediated by an immune response which comprises administering to said animal an antibody directed at a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA-1, CD80, CD86 and ICAM-1, preferably an anti-CD4 antibody, and a non-cellular protein antigen to generate a population of regulatory T-lymphocytes which are then re-activated by subsequent administration of the original non-cellular antigen.
  • a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA-1, CD80, CD86 and ICAM-1, preferably an anti-CD4 antibody, and a non-cellular protein antigen to generate a population of regulatory T-lymphocytes which are then re-activated by subsequent administration of the original non-cellular antigen.
  • the condition mediated by an immune response is generally an autoimmune condition.
  • autoimmune conditions include rheumatoid arthritis, multiple sclerosis insulin-dependent diabetes melitus and inflammatory bowel disease.
  • CD4+ T cells play a central role in autoi munity and have the capacity to be both protective and pathogenic. Accumulating evidence suggests that autoimmunity probably results when normal regulatory functions of protective CD4+ T cells break down. Autoimmune diseases can be treated to a certain extent by manipulation of CD4+ T cells. However, the effects may be only transient due to T cell turn-over and re-acquisition of T cell function.
  • T cells re-encounter auto-antigens that initiated the initial disease during on-going inflammation of the target tissue (for example the synovial joint in rheumatoid arthritis, pancreatic ⁇ -cells in insulin-dependent diabetes) the T cells will become activated and autoimmune destruction will re-occur. It may be possible to re-establish a balance between pathogenic and protective T cells by transient therapy designed to disable/deplete activated T cells followed by the administration of a non-cellular protein antigen such as HGG combined with additional or adjunctive immunotherapy using for example anti-CD4 antibody.
  • a non-cellular protein antigen such as HGG combined with additional or adjunctive immunotherapy using for example anti-CD4 antibody.
  • Regulatory T cells generated in this way would be reactivated by further administration of the non-cellular protein antigen and these might migrate to sites of inflammation along chemokine gradients and could arrive in the joints ready to suppress autoreactive T cells.
  • a protocol such as this would involve firstly depletion or inactivation of auto-reactive cells, secondly administration of non-cellular protein antigen together with monoclonal antibody immunotherapy and thirdly, reactivation of putative regulatory cells by subsequent administration of repeated doses of the original non-cellular antigen.
  • regulatory T cells generated ex vivo by exposing CD4 + T cells in culture to cells presenting donor alloantigen or an unrelated protein antigen in the presence of an antibody directed at a cell surface antigen selected from the group consisting of CD4, CD8, CD154, LFA-1, CD80, CD86 and ICAM-1, preferably an anti-CD4 antibody, could be used either as an alternative or complementary therapeutic approach to treat the condition mediated by an immune response.
  • T cells generated in the cultures would be administered intravenously to the animal.
  • Alloantigen-induced T-regs control the rejection of donor-specific skin grafts
  • CD25 + CD4 + cells were isolated from CBA (H2 k ) mice pre-treated with the anti- CD4 antibody YTS 177 together with blood from either B 10 (H2 b ) or BALB (H2 d ) mice and transferred them into syngeneic immunodeficient CBA-Rag _ " mice together with CD45RB hlgh CD4 + effector cells. One day later these recipients were transplanted with skin grafts either matched to the blood transfusion donor or from a third party strain ( Figure la).
  • Regulatory T cells generated in vitro by pre-culturing CD4 + T cells with anti-CD4 monoclonal antibody CmAb can control the rejection of donor-specific skin grafts
  • CD4 + T cells from naive CBA mice were cultured with irradiated antigen presenting cells from C57B1/10 mice in the presence of 5 ⁇ g/ml of 177 YTS anti-CD4 antibody.
  • After 8 days in culture total CD4 + or CD62L + CD25 + CD4 + CBA T cells pre-cultured with anti-CD4 mAb were transferred into syngeneic immunodeficient CB A-Rag " " mice together with CD45RB high CD4 + effector cells.
  • T-regs generated against unrelated antigen can regulate skin allograft rejection
  • T-reg specific for a non-cellular protein antigen could offer a potential tolerance induction strategy in man if such cells could regulate responses to alloantigens in vivo once re-activated. It has previously been demonstrated that tolerance to soluble antigens such as albumin 34 or human gamma globulin (HGG) 35,36 can be achieved by intravenous administration to mice under the cover of anti-CD4 antibody. We therefore chose HGG as a candidate for the tolerising antigen for these experiments. First, in order to determine whether induction of tolerance to HGG could be replicated in our hands we administered YTS 177 and HGG and then measured serum anti-HGG antibody concentrations by ELISA ( Figure 3d).
  • mice that received a priming protocol (where HGG was given without YTS 177) produced high levels of anti-HGG antibody whereas mice given HGG under the cover of YTS 177 gave low antibody titers identical to those from unprimed naive mice, indicating tolerance to HGG.
  • mice pre-treated according to the tolerising plus re-activation protocol antigen under the cover of YTS 177 followed by a second dose of antigen the day prior to analysis
  • mice with HGG following 177/HGG pre-treatment leads to JJFN- ⁇ mRNA production
  • mice were pretreated with the 177/HGG + HGG re-boost protocol and in addition received anti-CD 8 at the time of transplant.
  • Three out of four mice in this group accepted their C57BL/10 cardiac allografts for greater than 100 days ( Figure 5).
  • FIG. 1 Alloantigen-induced CD25 + CD4 + cells can regulate skin allograft rejection a, Pre-treatment and adoptive transfer protocol.
  • CBA mice were pre-treated with YTS177 on days -28 and -27 together with allogeneic (B10 or BALB) blood transfusion on day -27).
  • B10 or BALB allogeneic
  • CD25 + CD4 + cells from the spleens of these animals were adoptively transferred into CBA-Rag "7" recipients together with CD45RB hlgh CD4 + cells from na ⁇ ve animals, and the following day a B10 or BALB skin allograft was performed,
  • FIG. 2 T cells generated in vitro by co-culturing CD4+ T cells with cells presenting donor alloantigens can regulate skin allograft rejection a, Effect of cells generated in vitro by co-culturing CD4 T cells with cells presenting donor alloantigens and anti-CD4 monoclonal antibody on CD45RB hlgh CD4 + -mediated skin allograft rejection.
  • FIG. 3 Activated CD25 + CD4 + cells generated against unrelated antigen can regulate skin allograft rejection a, Induction of tolerance to HGG.
  • CBA mice were pre-treated as follows and serum anti-HGG antibody titer was measured by ELISA: A: naive; •: tolerizing protocol - YTS 177 on days -42, -41, and -40, and HGG on days -41, -14, and -7; ⁇ : priming protocol - YTS 177 on days -42, -41, and -40, and HGG on days -14 and -7; ⁇ : protocol used for in vivo adoptive transfer - YTS 177 on days -28 and -27 and HGG on days -27 and -1.
  • CBA mice were pre-treated with YTS 177 on days -28 and -27 and HGG on day -27.
  • Figure 6 Proposed models for regulation of allograft rejection by regulatory cells
  • Rejection may be overcome by the generation of regulatory populations specific for all of the alloantigens expressed on a graft (full repertoire).
  • regulatory populations may be generated against a single graft antigen; these regulatory cells undergo activation by the graft and then suppress responses against other graft antigens (bystander regulation).
  • regulatory cells generated against third party or even completely unrelated antigen can suppress graft rejection by bystander regulation provided that they are first activated before their functional activity is tested.
  • the hybridoma TIB 120 (anti-MHC class II) was obtained from American Type Culture Collection (ATCC), Manassas, VA, U.S.A.; YTS169 (anti- CD8) and YTS 177.9 (anti-CD4) 36 were kindly provided by Professor H. Waldmann (Sir William Dunn School of Pathology, Oxford, U.K.).
  • RM4-5 anti-CD4)-CyC, 16A (anti-CD45RB)-PE, 7D4 (anti-CD25)-biotin, and streptavidin-PE were purchased from Pharmingen (San Diego, California, U.S.A.).
  • IB 1.2 a blocking rat IgGl antibody reactive with mouse IL-10R (ref. 42).
  • GL113 a rat IgGl isotype control antibody reactive with ⁇ -galactosidase 43 .
  • Human gamma globulin was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.) and was heat aggregated at 63°C for 25 minutes and then incubated overnight on ice prior to use.
  • CD4 + T cells from na ⁇ ve CBA mice were cultured at 2xl0 5 cells per well in the presence of 5 ⁇ g/ml of YTS 177 anti-CD4 monoclonal antibody together with 5x10 5 irradiated (3600 rad) allogeneic total splenocytes per well from B10 mice.
  • Culture medium was composed of RPMI 1640 supplemented with 10% FCS (both P. A. A. Laboratories GmBH, Linz, Austria), 2mM L-Glutamine, 0.5mM 2-mercaptoethanol (Sigma, St Louis, Missouri, U.S.A.), and 100 units/ml each penicillin, streptomycin and kanamycin. All cultures were set in U shaped 96 well plates (Corning Costar, Cambridge, MA). Cells were kept in culture for 8 days at 37°C, 5% CO 2 and then harvested.
  • CD4 + (in vitro cultures) and CD45RB hlgh CD4 + (in vivo adoptive transfer experiments) T cells were isolated from lymph nodes and spleens of naive CBA mice, and CD25 + CD4 + T cells were obtained from spleens of animals pre-treated with YTS 177 and allogeneic blood or HGG.
  • CD25 + CD4 + and CD62L + CD25 + CD4 + T cells were obtained from in vitro co-cultures of CD4 T cells with cells presenting donor alloantigens and anti-CD4 monoclonal antibody (YTS 177). Populations were purified by negative selection using magnetic beads followed by FACS sorting as previously described. On re-analysis, all populations were >95% pure.
  • CBA-Rag "7" mice were reconstituted intravenously with 10 5 CD45RB high CD4 + cells with or without 2xl0 5 CD25 + CD4 + cells.
  • the following day full thickness BIO or BALB tail skin allografts were transplanted onto graft beds prepared on the flanks of the reconstituted mice.
  • anti-ILlOR antibody or isotype control
  • Allografts were monitored and graft survival between groups was compared using the log ranlc test 44 using software developed and kindly provided by Dr. S. Cobbold, Sir William Dunn School of Pathology, Oxford, U.K.
  • Skin grafts were fixed in buffered 10% formalin. 6 ⁇ m paraffin-embedded sections were cut and stained with hematoxylin and eosin.
  • Serum concentration of anti-HGG antibodies was measured by ELISA using a modification of standard methods. Plate-bound HGG was used to capture serum anti- HGG antibody which was then revealed and quantified using horseradish peroxidase- conjugated rabbit anti-mouse IgG and IgM (Jackson JmmunoResearch Laboratories, West Grove, PA, U.S.A.) followed by ABTS (2,2'-azino-bis(2-ethyl-benzathiazoline-6- sulfonic) acid) 45 . Absorbance at 405 nm was read and results are presented as the mean of duplicate wells ⁇ standard deviation.
  • C57BL/10 cardiac allografts were transplanted into the abdomen of CBA recipients as described 28 . Briefly, the donor aorta and pulmonary artery were anastomosed end-to side to the recipient descending aorta and inferior vena cava respectively. Graft function was followed by abdominal palpation and graft function at 100 days post transplant confirmed by laparotomy and direct visual inspection.
  • CD8 - T cell-mediated graft rejection implications for anti-CD154 immunotherapy. J. Immunol. 169, 5401-5404 (2002).

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Abstract

On peut supprimer le rejet des transplants chez l'animal: en administrant un anticorps dirigé contre un antigène de la surface cellulaire choisi parmi CD4, CD8, CD154, LFA-1, CD80, CD86 et ICAM-1, et de préférence l'anticorps anti CD4, et un anticorps dirigé contre un antigène protéique non cellulaire, de manière à produire dans l'animal une population de lymphocytes T régulateurs, et à transplanter l'organe ou le tissu alors que la population de lymphocytes T régulateurs est activée. On peut produire les lymphocytes T régulateurs ex vivo en cultivant des lymphocytes T avec un anticorps dirigé contre un antigène de la surface cellulaire choisi parmi CD4, CD8, CD154, LFA-1, CD80, CD86 et ICAM-1, en présence de cellules présentant soit un alloantigène, soit un antigène protéique non cellulaire. Les lymphocytes T produits ex vivo peuvent également servir pour surmonter le rejet des transplants, seuls ou associés au procédé in vivo. Une approche similaire peut être adoptée pour le traitement d'états auto-immunes.
EP04743000A 2003-06-20 2004-06-18 Suppression du rejet des transplants Withdrawn EP1639012A2 (fr)

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GB0608054D0 (en) * 2006-04-24 2006-05-31 Isis Innovation Production and use of regulatory t cells
EP2265643B1 (fr) 2008-03-13 2016-10-19 Biotest AG Schema posologique pour traiter le psoriasis et l'arhtrite rhumatoide
EP2260057B1 (fr) 2008-03-13 2016-11-23 Biotest AG Régime posologique d'un anticorps anti-CD4 pour le traitement de maladies auto-immunes
RU2540013C2 (ru) 2008-03-13 2015-01-27 Биотест Аг Средство для лечения заболевания
WO2009149382A2 (fr) * 2008-06-06 2009-12-10 Baylor Research Institute Anticorps anti-cd8 bloquant l’amorçage d’effecteurs cytotoxiques et conduisant à la génération de cellules t cd8<sp>+</sp> régulatrices
BRPI0919489A2 (pt) * 2008-09-29 2015-12-01 Biotest Ag composição, kit, métodos de tratamento de uma doença reumática, e de artrite reumatóide em um paciente, agente capaz de ativar células t reguladoras cd4+cd25+ e metotrexato, e, uso de um agente capaz de ativar células t reguladoras cd4+cd25+ e metotrexato
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EP2699263A4 (fr) * 2011-04-20 2014-12-24 Liquidating Trust Procédés pour réduire une réponse immunitaire indésirable à un antigène étranger chez un sujet humain avec des anticorps anti-cd4 ou des fragments de ceux-ci se liant aux cd4 ou des molécules se liant aux cd4
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